Capillary electrophoresis is superior to high performance liquid chromatography in that the amount of sample required for analysis can be less that 1/1,000 of the amount required for liquid chromatography and in that the separation performance as compared with liquid chromatography is tens of times higher. Capillary electrophoresis can be conducted in a shorter period of time than that for conventional slab gel electrophoresis and the like, because a higher voltage can be applied. Furthermore, when one end of the capillary is used as an injector, the samples can be injected automatically to provide an automated capillary electrophoresis apparatus. Capillary electrophoresis is described, for example, in Science, 222, 266-272 (1983). DNA sequencing is described in Analytical Chemistry, 62, 900-903 (1990).
Recently, capillary electrophoresis has been actively applied to many fields. In particular, nucleic acids and proteins have been analyzed as part of the recent development in biotechnological studies. In order to detect nucleic acid and proteins, ultraviolet detection and fluorescent detection have been used. For such detection, an on-column detection device that is integrated with the capillary is commonly employed.
In the analysis of nucleic acids, and particularly in DNA sequencing, the capillary electrophoresis is usually performed using a polyacrylamide gel. By this method, approximately 300 bases can be identified within about 45 minutes. Although it has been reported that DNA fragments consisting of about 10,000 bases have been separated within 20 minutes, the resolution was in the order of 1,000 bases in the case of DNA fragments consisting of thousands of bases. The analysis of DNA fragments consisting of about 10,000 bases has been described in Journal of Chromatography, 516, 33-48 (1990).
In the conventional on-column detection method with the use of a detector integrated with a capillary, the detection was performed by providing an optical path in part of the capillary. In general, an agarose gel was used as the slab gel electrophoresis for separating DNA consisting of 1,000 or more bases. In order to separate DNA having more bases than this and up to thousands of bases, it is necessary to use an agarose gel, rather than a polyacrylamide gel. However, an agarose gel has a poor light transmittance characteristic and is therefore unsuitable for on-column detection. As a result, it has been highly difficult to separate DNAs consisting of thousands of bases by capillary electrophoresis. Further, an agarose gel has heat resistance problems so it has been difficult to use such a gel in capillary gel electrophoresis where a high voltage is applied and Joule heating is created.
Genetic polymorphism, which is useful in gene diagnosis and gene tests required for medical purposes, relies on polymorphisms built up in human genes and other animals that have medical information relating to genepathy and immunological mechanisms. For example, more than 400 polymorphisms are built up in a histocompatibility antigen (HLA) gene, which closely relates to an immunological mechanism and combinations of these polymorphisms give more than 1 or 10,000 serotypes. It is particularly important in, for example, organ transplantation to determine these serotypes. Further, there is suggested a relationship between these serotypes and various diseases. Thus, the determination of HLA serotypes will become highly important in the future and a method for quickly and accurately analyzing the polymorphism is very important in the fields of diagnosis and medical examinations.
The detection of genetic polymorphism on a monobase level means the detection of a mutation in a gene arrangement and relates to the diagnosis of a number of diseases including cancer. It is suggested that gene mutation would relate to the mechanism of carcinogenesis or metastatis. Therefore, the rapid classification and determination of genetic polymorphisms and mutations of a number of specimens, if possible, will contribute greatly to the diagnosis and identification of carcinoma.
To detect genetic polymorphism on a monobase level with high sensitivity, it has been proposed, for example, by M. Orita et al in the Proceeding of National Academy of Science of U.S.A., 86, 2766-2770 (1989) to analyze the base sequence polymorphism of a DNA by using a difference in higher-order structure between single-strand DNAs (single strand conformation polymorphism, hereinafter referred to as the SSCP method). This method includes disassociating the target region on a DNA base sequence, which has been extracted by an appropriate method, into a pair of single-strand DNAs which are complementary to each other by an appropriate denaturing means and then electrophoresing these single-strand DNAs on an undenatured polyacrylamide gel. In this procedure, the migration rate of each single-strand DNA varies under the influence of the higher-order structure of the single-strand DNA and this higher-order structure is specifically determined depending on the sequence of the single-strand DNA. Thus, a sequence polymorphism differing in at least one base can be detected by taking advantage of the above-mentioned property.
Since this method of SSCP is highly convenient and enables the detection of a polymorphism due to a difference in a single base at a high sensitivity, it is thought to be quite advantageous as compared with the restriction fragment length polymorphism method and the other well known conventional method for analyzing polymorphism that uses a DNA probe specific for a sequence. In recent years, the SSCP method has been combined with the polymerase chain reaction method (PCR method) in the analysis of polymorphism or the identification of gene mutation.
It has been difficult to achieve a high separation performance and to effect quantitative analysis of electrophoretic patterns, which provide valuable information for analyzing the polymorphism, by the conventional SSCP method, wherein the electrophoresis is performed by using a slab gel. Further, additional problems exist when this method is performed, mainly the migration and detection require a long time, the denatured sample of the single-strand DNAs undergo reassociation during the procedure of packing them in a gel, and it is difficult to control the temperature during the migration which greatly affects the maintenance of the higher-order structures. Under these circumstances, one object of the present invention is to provide a method for analyzing genetic polymorphism by the SSCP method whereby the analysis can be conveniently completed within a short period of time to achieve a high separation as compared with the conventional methods. The invention further aims at enabling automatic analysis of polymorphism by the SSCP method.
Conventionally, a gel filled capillary has been produced by packing an acrylamide solution containing a polymerizing agent into a capillary by using, for example, a syringe and them effecting gelling polymerization in the capillary. However, this method suffers from a problem that air bubbles are formed in the polymerization of the acrylamide and thus the acrylamide gel thus formed is practically unusable. To counteract this problem, a method has been employed wherein the acrylamide is polymerized under a high hydraulic pressure and also wherein a carefully degassed acrylamide solution is treated by suction or vacuum and packed into the capillary. U.S. Pat. No. 4,810,456 discloses a method for polymerizing acrylamide under high hydraulic pressure. A method for vacuum injection of a degassed acrylamide solution is disclosed in Analytical Chemistry, 64, 1221-1225 (1992).
The production of a capillary gel by polymerizing acrylamide under a high hydraulic pressure has been performed by packing an acrylamide solution containing a polymerizing agent in a capillary by using, for example, a syringe and subjecting the capillary packed with the acrylamide solution to gelling polymerization under a high hydraulic pressure. This method suffers from a problem that the acrylamide solution is diluted before the polymerization of the acrylamide, and thus it is difficult to form a gel with high reproducibility of the result. In addition, the load in the capillary increases as the inner diameter of the capillary decreases, and, as a result, the injection becomes difficult. Accordingly, most of the capillaries usable in this method have an inner diameter of at least as great as 75 .mu.m and there have been only a few reported examples of using capillaries having an inner diameter of 50 .mu.m. Although it is possible to use, for example, a pump for packing the acrylamide solution, a long channel from the solution tank to the capillary is inevitably required when a pump is employed, and therefore, a large amount of the acrylamide solution is required. Furthermore, a considerably long period of time elapses during feeding the solution from the pump to the capillary, which causes another problem that the polymerizing agent added before the injection causes the acrylamide to gel in the flow channel.
With the other conventional method involving the vacuum or suction injection of a carefully degassed acrylamide solution and packing thereof in a capillary, there exists a problem that the vacuum injection of the acrylamide solution makes the pressure in the capillary negative, thus creating a potential for air bubbles to be formed if the degassing is insufficient.
Conventional methods of injecting a sample include gravity injection, which relies upon the difference in gravity, pressure injection, which relies upon the use of a difference in pressure and electrokinetic injection. The injection method affects the analytical accuracy and reproducibility of results with capillary electrophoresis. With these conventional methods, control is difficult since the difference in gravity or pressure is difficult to control when a minute amount of the sample is injected. In particular, a trace amount of a sample is difficult to inject by the methods of gravity injection and pressure injection. In the electrokinetic injection, on the other hand, the amount of sample to be injected is determined by the amount and duration of the applied voltage. Accordingly, a trace amount of a sample can be readily injected with high accuracy by controlling the amount and duration of the applied voltage. As a result, the electrokinetic injection method is the most commonly employed method at the present time. This method, however, suffers from a problem that the amount of a sample to be injected varies depending on the components contained therein, since the mobility varies from substance to substance.
One solution proposed for this problem has been set forth in Japanese Patent Laid-Open Application No. 253247/1988, wherein it has been proposed to meter a sample with a rotary injector and inject the sample under pressure. Specifically, the sample is sandwiched between a gel and a buffer during the step of injecting the sample. As a result, the sample is diluted with the buffer before being incorporated into the gel by pressure injection with the result that the sensitivity and separation performance are degraded due to an increase in the widening of bands caused by, for example, disorders in the band.